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Agarose Co-IP and Pull-Down Kits
Pull-down is an extension of co-immunoprecipitation (Co-IP) for the capture and isolation of a target protein (i.e., the antigen) as well as its associated partners. Tag-based IP and pull-down strategies utilize a "bait" protein instead of an antibody to capture protein-interaction targets by utilizing an affinity resin to the bait protein that is biotinylated his-, GST-, c-Myc or HA-tagged.
- More choices—different strategies to accommodate various labeled bait proteins, elution conditions, and downstream analysis needs
- Complete—kits include all controls and lysis, binding, wash, and elution buffers
- Fast—spin columns and collection tubes are included that shorten the protocol and minimize sample handling
- Flexible—can scale up or down as needed using adaptable protocol
 |  |  |  |  | | |
|---|---|---|---|---|---|---|
| Pierce Biotinylated Protein Interaction Pull-Down Kit | EZ-Link Desthiobio-tinylation and Pull-Down Kit | Pierce c-Myc Tag IP/Co-IP Kit | Pierce HA Tag IP/Co-IP Kit | Pierce GST Protein Interaction Pull-Down Kit | Pierce His Protein Interaction Pull-Down Kit | |
| Target | Biotinylated proteins | Biotinylated proteins | c-Myc–tagged recombinant protein | HA-tagged recombinant protein | GST-tagged recombinant protein | His-tagged recombinant protein |
| Base bead | Streptavidin agarose resin, | High-capacity streptavidin agarose resin | Anti-c-Myc agarose resin | Anti-HA agarose | Glutathione agarose | Cobalt chelate agarose resin |
| Binding capacity | 1–3 mg/mL | >10 mg/mL | 102 nmol/mL | >60 nmol/mL | 8–10 mg/mL | 10–25 mg/mL |
| Non-specific binding | Low | Low | Lower | Lower | Lowest | Lowest |
| Elution conditions | Low pH (2.8) Buffer | Mild (free biotin buffer) | Low pH (2.0) buffer | Non-reducing sample buffer | 100 mM reduced glutathione | 250 mM imidazole solution |
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Effectiveness of elution options in the IP of GST-HA and GST-c-Myc from bacterial lysates.
IP results achieved with the Thermo Scientific Pierce HA IP/Co-IP Kit and c-Myc IP/Co-IP Kit using the appropriate positive control lysate provided and suggested elution options for GST-HA and GST-cMyc, respectively. The elution components are supplied with each kit. Elution #1 performed with elution buffer; Elution #2 performed with 2X nonreducing sample buffer.
| Lane # | A. GST-Tag Pull-Down | B. PolyHis-Tag Pull-Down |
|---|---|---|
| 1 | Lysate from E. coli expressing GST-tagged BIR2 (bait protein). | Lysate from E. coli expressing 9xHis-tagged wild-type Smac (bait protein). |
| 2 | Flow-through from the lysate in Lane 1 bound to an immobilized reduced glutathione support for 1 hour at 4°C. | Flow-through from the lysate in Lane 1 bound to an immobilized cobalt chelate support for 1 hour at 4°C. |
| 3 | Wash #1 of the support. | Wash #1 of the support. |
| 4 | Wash #2 of the support. (Washes 3-5 not shown.) | Wash #2 of the support. (Washes 3-5 not shown.) |
| 5 | Lysate from E. coli expressing 9xHis-tagged wild-type Smac (prey protein). | Lysate from E. coli expressing GST-tagged BIR2 (prey protein). |
| 6 | Flow-through from the lysate in Lane 5 is allowed to interact with the immobilized bait for 1 hour at 4°C. | Flow-through from the lysate in Lane 5 is allowed to interact with the immobilized bait for 1 hour at 4°C. |
| 7 | Wash #1 of the support. | Wash #1 of the support. |
| 8 | Wash #2 of the support. (Washes 3-5 not shown.) | Wash #2 of the support. (Washes 3-5 not shown.) |
| 9 | Bait control. Bait treated as described in Lanes 1-8 and subsequently eluted. No prey added – just binding buffer. | Bait control. Bait treated as described in Lanes 1-8 and subsequently eluted. No prey added – just binding buffer. |
| 10 | Prey control. Prey treated as described in Lanes 1-8 and subsequently eluted. No bait added – just binding buffer. | Prey control. Prey treated as described in Lanes 1-8 and subsequently eluted. No bait added – just binding buffer. |
| 11 | Elution of bait:prey complex (prepared in Lanes 1-8) from the support with 100 mM reduced glutathione. Western blotting confirms that the minor bands observed in Lanes 9 and 11 are degradation products of GST-tagged BIR2. | Elution of bait:prey complex (prepared in Lanes 1-8) from the support with 250 mM imidazole. |
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For Research Use Only. Not for use in diagnostic procedures.